Numerous investigators in the selenium field have proposed that low molecular weight selenocompounds are responsible for the numerous health benefits attributed to selenium, while others have suggested that selenoproteins are likely responsible. These health benefits include preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process and having roles in mammalian development, male reproduction and immune function. We proposed several years ago that these health benefits are due largely to the presence of selenium in selenoproteins as the amino acid, selenocysteine (Sec). Therefore, to elucidate the role of selenoproteins in cancer prevention and development, we are characterizing the function of several selenoproteins. During the past year, we have focused our attention on studying in greater detail the role of three selenoproteins, thioredoxin peroxidase 1 (TR1), glutathione peroxidase 4 (GPx4) and selenoprotein 15 (Sep15). We had previously shown that the knockdown of TR1 using RNA interference technology in a lung cancer cell line (JBC 281: 13005, 2006) and in a cancer cell line driven by oncogenic k-ras (PLoS One 2: e1112;1-7, 2007) resulted in several of the malignant phenotypes being reversed more towards those of normal cells suggesting that TR1 deficiency is antitumorigenic. This past year, we have continued to focus on the molecular basis of TR1s role in cancer development and have examined the role of TR1 in apoptosis in a breast cancer cell line in detail. The breast cancer cell line was selected since we found that TR1 knockdown in breast cancer cells showed a much higher sensitivity to TNF-alpha induced apoptosis. We are currently confirming these findings in mice in vivo as further discussed in Project 1 (Z01 BC 005317). We are also continuing our study on the intracellular role of GPx4 and have found GPx4 is essential for repairing membrane lipid hydroperoxidation. Furthermore, we have evidence that this selenoprotein may be involved in the etiology of neurodegenerative diseases such as Alzheimers and Parkinsons diseases. GPx4 has decreased levels in brain tissue of Alzheimers and Parkinsons disease mouse models. GPx4 is a selenium-containing, antioxidant enzyme whose intracellular function has not been fully resolved. To elucidate its function intracellularly, we targeted the removal of GPx4 in a mouse fibroblast cell line (NIH3T3 cells) that resulted in severe damage to the cells when the level of this enzyme was reduced below 80% of its normal level. Partial reduction in its expression led to severe growth retardation. Unexpectedly, GPx4 knockdown cells showed little change in levels of reactive oxygen species, but these cells manifested highly increased levels of oxidized lipid byproducts suggesting membrane lipid hydroperoxidation damage. The data thus far have established an essential role of GPx4 in the metabolism of membrane lipid hydroperoxides, and surprisingly, a limited role as a general antioxidant enzyme. In the past year, we have found that Sep15 appears to have a role in colorectal cancer. Our findings demonstrate that knocking down the expression of Sep15 in the murine colon carcinoma cell line, CT-26, using RNAi resulted in phenotypic changes more towards normal cells that included decreased cell growth rates and reduced anchorage-independent growth abilities. Furthermore, CT-26, Sep15 knockdown cells, when injected into mice of the same background and compared to plasmid-transfected controls, also demonstrated a significantly reduced ability to form localized subcutaneous tumors and lung metastases in vivo. Mouse Lewis lung carcinoma cells (LLC1), transfected with the same siSep15 construct, appeared to maintain their original growth characteristics, indicating that knockdown of Sep15 reduced tumorigenicity of colon (CT26), but not lung (LLC1) cells. We are continuing to investigate this apparent tissue specificity, as well as possible functions of Sep15 in colon and other tissues.